Epigenetics is the discipline where information inherited or succeeded to after cell division is handled without being based on the base sequences of genes. This information is maintained by DNA methylation and histone modifications. DNA methylation or histone modifications mainly control gene switching, i.e., whether or not genes work. For this reason, cells and individuals, even with exactly the same gene base sequences, can have totally different phenotypes.
DNA methylation occurs mainly as the methylation of the 5-position carbon in the cytosine residue; for example, in higher eukaryotes, the cytosine present in the CpG sequence (a sequence with cytosine (C) followed by guanine (G), from the 5′ side, in the 5′-CG-3′ arrangement on the base sequence of DNA) is known to be methylated. The CpG sequence is also present in promoter regions of many genes; it is generally said that the methylation of the cytosine in the CpG sequence in the promoter region suppresses gene transcription. For this reason, it is thought that the methylation of the cytosine in the CpG sequence represents an important epigenetic mechanism involved in the regulation of gene expression in higher eukaryotes, and plays an important role in the cellular functions themselves.
Epigenetics is involved in a wide variety of life phenomena such as development, differentiation, genome imprinting, and X chromosome inactivation. Also, in diseases, methylation abnormalities in cancer are well known; it has recently been suggested that methylation abnormalities are also involved in a wide variety of non-cancer diseases such as schizophrenia and diabetes. Also, it has been increasingly evident that the methylation is instable in vitro; it is thought that it will become important in regenerative medicine to determine whether the methylation state of organs differentiation-induced from stem cells is normal.
While 60 to 90% of cytosine residues in the CpG sequence are methylated in mammals, the cytosine in the CpG sequence (CpG island), which is densely present in promoter regions of genes, is often not methylated (non-patent document 1). However, if an unmethylated region is methylated by a certain cause, gene transcription will be suppressed. For example, if the transcription of a cancer suppressor gene in cancer cells is inactivated, the growth of the cancer cells will become uncontrollable. Conversely, if the CpG island, which is normally methylated, fails to be methylated due to a certain abnormality, gene inactivation (stability) cannot be maintained, which in turn not only prevents the cells from exhibiting their essential functions, but also can produce functional abnormalities such as cell and tissue differentiation abnormalities. Therefore, the presence or absence of methylation and changes in the pattern thereof can provoke diseases such as cancer and differentiation abnormalities; examining the methylation pattern is strongly demanded also for the sake of treatment and prevention of such diseases.
To detect methylated DNA, a wide variety of methods have traditionally been attempted; for example, a method using a methylation-sensitive restriction endonuclease is known (non-patent documents 2 to 7). A methylation-sensitive restriction endonuclease is an enzyme that is unable to cleave DNA when the recognition site is methylated; although this method is effective in detecting methylation, the CpG site to be analyzed need to be a recognition sequence for the methylation-sensitive restriction endonuclease, so that the degree of freedom of the analysis is low, and a pretreatment for DNA purification is needed. Also, an analytical time of several hours or more to detection is required, including enzymatic reaction time.
Against this background, as methods of detection not relying on nucleotide sequences, methods wherein unmethylated cytosine is reacted with a hydrogen sulfite salt to convert it to uracil, and this is followed by PCR and sequencing (non-patent documents 8 to 13), immunoprecipitation methods using an anti-methylated cytosine antibody have been developed (patent document 1, non-patent documents 14 to 20). However, these methods also necessitate pretreatments such as alkali treatment and fragmentation; a long detection time of 5 to 16 hours is taken, with no improvement achieved in detectability at high sensitivity. In contrast, methods of specifically detecting methylated cytosine by allowing a combination of bipyridine-modified DNA and osmium oxide to selectively form a complex with methylated cytosine have been developed (non-patent documents 21 to 25), enabling the detection at higher sensitivity thanks to signal intensification, but the drawback of necessity for pretreatments such as heating after complex formation could not be resolved. Also, attempts have been made for methods utilizing methylated DNA-binding protein (MBP) (patent documents 2 and 3), but the molecular weight of MBP itself is extremely high at several tens of thousands to several hundreds of thousands, and there is still room for improvements in terms of stable supply, storage stability, or the ease of handling.